Method for fixing and embedding tissues for histological preparations

ABSTRACT

The invention concerns a method whereby a tissue is fixed by a liquid fixative, dehydrated by a liquid dehydrating agent, and infiltrated/embedded in a molten compound having a melting point higher than room temperature, wherein the method comprises the steps of fixing the tissue in a liquid fixative comprising at least a soluble zinc salt, in an aqueous buffer solution, dehydrating the fixed tissue in a liquid consisting essentially of acetone, embedding and infiltrating the dehydrated fixed tissue with a resin essentially soluble in acetone.

BACKGROUND OF THE INVENTION

The invention concerns a method for fixing and embedding tissues forhistological preparations.

DESCRIPTION OF RELATED ART

Two types of methods are currently used in this field, namely

1. slicing of frozen sections

2. slicing of fixed and embedded tissue sections.

Slicing of Frozen Sections

Freezing fulfils the dual function of fixing the tissues in thecondition in which they are found and to solidify them to enable them tobe sliced.

Slicing of frozen samples provides a histological material:

which exhibits substantially no molecular denaturation and thus enablesgood determination with respect to the nature of the molecules present

which has the disadvantage that it appears in the form of thicksections, thus not enabling a microscopic examination at greatmagnification, and in which the morphological structure of the tissuehas been greatly altered by the freezing

such that, if it is possible to label the molecules, it is not possibleto reliably locate the molecules thus labelled in the morphologicalstructure of the tissue.

Finally, the slices made in the frozen tissue are then prepared andexamined at room temperature, and thus a chemical fixing is stillnecessary after the cut.

Slicing of Fixed and Embedded Tissue Sections

In this method, the tissue is (1) first fixed, then (2) subjected to adehydration step and finally (3) subjected to an embedding step tosolidify the tissue and enable it to be cut, after which (4) the tissueis cut into fine lamellae, generally by a microtome. These lamellae aredisposed on object slides and (5) these preparations are finally rid ofthe embedding product and rehydrated prior to the histochemicaltreatment.

The purpose of the fixing step (1) is to prevent substantially anyphysicochemical alteration of the tissue, to maintain it in the initialstate in which it was collected and to thus permit determinations, inparticular, immunological ones.

The purpose of the infiltration/embedding step (3) is to solidify thetissue to make it possible to make the slices as thin as possible, thusenabling a more extensive examination.

The dehydration step (2) is an intermediate step whose purpose is toreplace the water present in the tissues by a compound which is itselfsubstituted—possibly with the use of solvents—by theinfiltration/embedding compound.

Step (4) provides the embedded, and thus solidified, tissue sections andstep (5) finally restores the tissue more or less to its initialcondition, permitting the desired analysis.

Various liquid fixatives are known for implementing the fixing step (1),such as compositions based on soluble zinc salts, various organiccompounds (see e.g. EP-A-0562877), as well as acetone (see e.g. U.S.Pat. No. 5,104,640).

Various compounds, such as e.g. ethanol, methanol, isopropanol andacetone are known for the dehydration step (2) (see e.g. the article byBeckstead cited below).

For the infiltration/embedding step (3), paraffin is conventionallycurrently used. The disadvantage of the use of paraffin is that it isweakly soluble and consequently it is only possible to eliminate it byresorting to strong solvents and that it only melts at temperatures of58-60° C. This is substantially higher than the physiologicaltemperature of tissues and thus denaturing for them.

Jay H. Beckstead, in The Journal of Histochemistry & Cryochemistry (“ASimple Technique for Preservation of Fixation-sensitive Antigens inParaffin-embedded Tissues”, Vol. 42, No. 8, pp. 1127-1134, 1994)describes a method for fixing/embedding using a zinc salt fixative,together with a paraffin embedding, and comparing it, on the one hand,with known paraffin embedding methods together with other fixatives and,on the other hand, with the freezing method.

This method consists of

fixing the tissues with soluble zinc salts in a buffer solution,

dehydrating the tissues thus fixed by increasing concentrations ofethanol/isopropanol,

eliminating the ethanol/isopropanol with xylene,

infiltrating/embedding with paraffin at 58-60° C.,

making the slices,

eliminating the paraffin with three xylene baths, followed by threeisopropanol baths, and rehydrating.

Acetone—which also acts as a dehydrating agent—is known for causing aperceptible morphological denaturation of the tissues. For this reason,researchers consider it to be inappropriate for fixing when one wishesto make morphological determinations, which is quite generally the casefor histological sections prepared for examination with a microscope.

Alcohols (ethanol, methanol, isopropanol, for example) are known for notperceptibly degrading morphological structures when they are used withcare. They are, therefore, conventionally used for the dehydration oftissues. On the other hand, they have a marked degradation effect onmolecular structures (by coagulation) and, consequently, they havedisadvantages when both the molecular and the morphological structuresare to be preserved, as is the case in immunological studies in situ andex vivo in immunohistochemistry.

On the other hand, alcohols have a very great dehydrating power and, toavoid altering the morphological structure, it is necessary to carry outthe dehydration by successive steps, with increasing alcoholconcentrations, which obviously hampers the procedure.

Having regard to the fact that paraffin is generally of low solubility,it is necessary to provide, between the dehydration step and theembedding step, an intermediate step to eliminate the dehydrating agentwith solvents such as toluene, xylene, benzene or the like, whichcreates denaturation problems for tissues, and toxicity, and adds stillanother step to the mode of operation.

Finally, impregnation with paraffin requires temperatures of ±60° C.,higher than the physiological temperature, and this exerts a denaturingeffect (thermal coagulation) on the molecular structures.

Moreover, the process for eliminating paraffin and rehydration of thepreparations must also be done with strong solvents which are notwithout disadvantages.

A method is also known (“Immunofluorescence Detection of F-actin on LowMelting Point Wax Sections from Plant Tissues” by Stanislav Vitha et al,in The Journal of Histochemistry & Cryochemistry, Vol. 45(1): 09-95,1997) that consists of

fixing the tissues successively in formaldehyde and zinc chloridesolutions, the latter step being carried out at 60° C.,

dehydrating the tissues with increasing ethanol concentrations,

infiltrating/embedding, in several steps, the tissues in a resinconsisting of 90% polyethylene glycol 400 distearate [which is alsoknown under the name polyoxyethylene bis(stearate)] and of 10%1-hexadecanol, at 35-57°,

making the slices, and

eliminating the resin with ethanol of technical grade, and rehydratingthe preparations.

BRIEF DESCRIPTION OF THE INVENTION

According to the invention, a method for fixing/embedding is to beprovided which does not perceptibly denature either the molecularstructure nor the morphological structure of the tissues, to enableanalysis that is as reliable and precise as possible. This is important,in particular, for immunological analysis in situ and ex vivo, in whichdeterminations and immunological analyses are to be made not only onbiological molecules extracted from their cellular and/or tissueenvironment, but also such molecules in this environment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C are photographs of tissue sections of a mousespleen, obtained by freezing;

FIGS. 2A and 2B are similar views to FIGS. 1A, 1B and 1C after tissuepreparation by another method; and

FIGS. 3A, 3B and 3C are similar views of a mouse spleen tissue sectionafter treatment according to the present invention.

DESCRIPTION OF THE INVENTION

Therefore, an object of the invention is to provide a method making itpossible to obtain sections of tissues which are not substantiallydenatured, neither from the molecular point of view, so as to enablequantitative immunological determinations, nor from the morphologicalpoint of view, to make it possible to realize these determinations inthe original cellular and/or tissue environment.

The methods known in the prior art do not enable one to obtain thisobject insofar as they denature either the molecular structure or themorphological structure, or even both.

According to the invention, this object is attained by a method forfixing and embedding tissue for histological preparations, it beingpossible to implement it more or less at the physiological temperature,which essentially comprises the steps consisting of:

fixing the tissue in a liquid fixative comprising at least one solublezinc salt, in an aqueous buffer solution,

dehydrating the tissue thus fixed in a liquid consisting essentially ofacetone,

embedding and infiltrating the fixed and dehydrated tissue with a resinsubstantially soluble in any proportion of acetone.

According to another feature of the invention, the resin melts at atemperature of not higher than 37-40° C.

According to an additional feature of the invention, the resin consistsof polyoxyethylene bis(stearate).

According to still another feature, the resin comprises, based of 100%by weight of the final composition, 0 to 20% by weight of a solidifyingcompound, comprising hexadecanol and/or diethylene glycol distearate,for the purpose of rendering it more solid at room temperature.

The invention also concerns a process for the preparation ofhistological sections by applying the method which comprises essentiallya flask of an aqueous solution buffered with one or more zinc salts anda quantity of a polyoxyethylene bis(stearate) resin composition mixedwith hexadecanol and/or diethylene glycol distearate, depending on thedesired temperature of use.

According to another feature of the invention, the case also comprisesan acetone flask.

The method of the invention is applicable in all fields of histology andhistochemistry and its implementation is extremely simple. It can becarried out at a temperature of 35-37° C., i.e. substantially thephysiological temperature of living tissue.

The polyoxyethylene bis(stearate) resin is already known from thearticle by S. Vitha et al.

This resin, the melting point of which is 37° C., makes it possible tomake the cuts under good conditions up to temperatures of 10 to 12° C.The purpose of adding hexadecanol is to make it more solid at roomtemperature, in order to make it possible to make slices at thetemperature in the laboratory.

According to the invention, it was also found that hexadecanol can alsobe replaced in whole or in part by diethylene glycol distearate whichhas substantially the same properties (almost inert vis-a-vis tissues,and a hardening effect on the resin).

An important feature of the method of the invention, in addition toembedding at “normal” temperature, is the association of a zinc-saltbased fixative with the acetone as dehydrating agent.

Acetone does not generally appear in histology treatises (MARTOJA R. &MARTOJA M., 1967, “Initiation aux Techniques de l'Histologie Animale”,ed. MASSON et Cie) as a fixative, even if it is occasionally used as adehydrating agent. This is justified by its low penetration speed intissues and the morphological disorders that it causes. Nevertheless, itis cited as a fixative in some cases when one is looking to preserveenzymatic activity in situ.

POLLARD et al (J. Histochem. Cytochm., 1987, 35 (11): 1329) states thatmolecules such as CD4 and CD8, markers of T-lymphocytes, can no more beevidenced on tissue sections fixed with acetone and embedded in paraffineven though they remain detectable on frozen sections treated withacetone for at least 30 minutes. This result appears to show thatacetone itself is not denaturing but that, together with an embedding inparaffin, it no longer permits the detection of these two markers.

According to the inventor's research, the use of acetone as a fixativeand dehydrating agent, and of polyoxyethylene bis(stearate) resin as anembedding environment improves the performances of fixing/embeddingmethods by permitting the use of a greater number of antibodies and byfacilitating the making of fine cuts. In spite of this improvement, thismethod still does not permit the detection of molecules such as CD3, CD4and CD8 (see Table 2), thus confirming the results of POLLARD et al.Moreover, a certain number of morphological deformations induced byacetone are observed: dilatation of the tissues, appearance ofartifactual intercellular spaces.

The combination according to the invention of first fixing tissues withan aqueous solution buffered with a zinc salt, and dehydrating withacetone, followed by infiltration/embedding with a resin such aspolyoxyethylene bis(stearate) completely miscible with acetone,suppresses these deficiencies.

In fact, not only the preservation of molecular tissues appearscomplete, but the fixing with zinc salts seems to ensure that thetissues have a morphological stabilization such that they then becomeresistant to deformations induced by acetone.

The subsequent embedding with a resin which is completely miscible withacetone avoids, on the other hand, the need to recover intermediarysolvents to eliminate the acetone prior to embedding; this sameadvantage is also found later, when it is necessary to eliminate theresin and rehydrate the tissue.

For a person skilled in the art who knows the method according to S.Vitha et al, as well as the properties of acetone, and who wishes towork at room temperature while simplifying the method to a maximum, anormal process would therefore have consisted of using acetone as afixative and dehydrating agent, thus obtaining a substantiallydenaturing method from the morphological point of view.

Surprisingly, the inventors discovered that the combination of fixingwith zinc salts followed by dehydrating with acetone did not lead to theperceptible molecular denaturing normally produced by acetone.

Without wishing to be limited to this explanation in any way, it seemsthat the physicochemical fixing produced by zinc salts is such that itresists a subsequent perceptible modification with the acetone.

Thus, another significant advantage brought about by acetone lies in theacetone being, on the other hand, miscible in any proportion in thepolyoxyethylene bis(stearate) resin, such that it is not necessary tohave recourse to strong solvents such as, for example, benzene, tolueneor xylene to enable the transition from the acetone to the resin, whichagain reduces denaturing, simplifies the entire method by reducing thenumber of steps involved and also prevents environmental and toxicityproblems that might be associated with the above solvents.

The method of the invention thus ensures a perfect synergy between thedifferent operative stages for the preparation of the histologicalsections, to attain the desired result, namely minimal molecular andmorphological denaturing, by a method which has been remarkablysimplified vis-&-vis the prior art.

Broad fields of application will be found in the preparation ofhistological sections with a view to immunological determinations insitu and ex vivo, by the quality of the sections that it permits.

A preferred field for the method according to the invention isimmunohistochemistry. Within sections obtained by the method, it is infact possible to detect numerous molecules such as membrane markerscharacteristic of cellular lines; activation markers reflecting thestate of cells; cytoplasmic proteins including cytokines orantiapoptotic proteins; glycosidic residues evidenced by lectines,fragments of nucleic acids revealed by hybridization in situ or by theTUNEL reaction characterizing apoptosis. This list is not in any wayrestrictive and given only by way of example.

Another field of application of the method according to the invention isthe replacement of conventional methods for preparing histologicalsections for observation under a microscope, by the overallsimplification and reliability that it permits.

Example of the Procedure for Preparing Sections

The following procedure is followed:

1. Isolation of tissue

2. Fixing: small fragments of tissue (5 mm) are immersed in the liquidfixative (several ml) with zinc salts previously cooled to 4° C. and aremaintained therein for 1 to 7 days at 4° C. The fixing is carried out insmall pill-type glass flasks. To prevent precipitation of the zincsalts, the liquid fixative is obtained by dissolving 5% of zinc salts(acetate and chloride, in equal parts) in distilled water. This solutionis then added to a tri-HCl buffer adjusted to pH 7.4, containing 0.1%calcium acetate (1 volume of zinc salt solution for 9 volumes oftri-HCl) so as to obtain a buffered solution of zinc salts at 0.5%.

3. Dehydration: the liquid fixative is eliminated and replacedimmediately by pure acetone (analytical grade); the fragments are lefttherein for 6 to 24 hours at 4° C.;

4. Impregnation and embedding: the fragments are transferred into a pillflask maintained at 37° C. containing the liquid resin. To ensure a goodimpregnation, the resin bath is changed three times, each bath lastingfrom 10 to 30 minutes. After the third bath, the fragments are placed ina mold of appropriate size (1 to 3 cm³) previously filled with liquidresin. Once immersed in the liquid resin, the fragments can be orientedthere, dependent on the subsequent requirements; if necessary, the resinis maintained liquid during this operation by immersing a metal spatulaheated with the flame. When the fragment is correctly oriented, theresin is allowed to cool and solidify to room temperature in a drylocation. After a night to two days, the solid block of resin can beremoved from the mold and prepared to be cut into fine sections.

5. The fine sections (3 to 5 μm) are formed by a sliding or rotarymicrotome at room temperature. Given the low melting temperature of theresin, it can become difficult to cut at temperatures above 22° C.

6. The sections are placed on microscope slides previously gelatinizedor treated with polylysine. The resin being hydroscopic, it is advisedagainst placing on a water bath. It is preferable to place them on adrop of albumenised water directly on the slide; they are spread out atroom temperature until the slide is completely dried.

7. The slides thus obtained can be preserved indefinitely until thehistochemical treatment.

Example of Immunohistochemical Treatment

Elimination of the resin in an acetone bath for 10 minutes

Rehydration of the sections in a PBS bath (saline phosphate buffered topH 7.4) for several seconds (can be extended for several dozen minutes,if necessary)

Inhibition of the endogenic pseudoperoxydases (optional step if theenzymatic revelation method makes use of an enzyme other thanperoxydase): incubation of the sections in H₂O₂ at 1 to 3% for 10 to 30minutes. This step should, by necessity, precede all the others in orderto be effective.

Saturation of the non-specific fixing sites by the “Blocking Reagent” ofthe firm Boehringer (Catalogue No. 1 096 176) dissolved in PBS at pH7.4; BSA (Bovine Serum Albumin) can also be used at 1% in PBS.

Washing in PBS

Incubation of the slides with specific biotinylated antibodies dilutedin the solution of the above saturating agent. The incubation conditionsof the antibodies depends on each antibody; by way of example, aconcentration of 10 μg/ml antibodies gives good results after incubationof one hour at room temperature.

Washing in PBS

Incubation of the sections with the biotinylated avidine-enzyme (ABC)complex; this complex exists in three different forms comprising, asenzyme, either the peroxydase, or the alkaline phosphatase, or even theoxydase glucose; the choice of enzyme is left to the user's discretion;the best results are obtained with the complexes marketed by the firmVECTOR

Washing in PBS

Revealing the enzymatic activity with aid of specific substrates,obtained from the firm VECTOR

Washing with running city water

Counter-coloration (depending on the substrate used) and assembly ofpreparations

The primary advantages of the invention are:

1. the extreme simplification of the fixing and embedding method;

2. the simultaneous preservation of the morphological and molecularstructures of the treated tissue.

Comparative examples will enable the illustration of the advantages ofpoint 2.

The currently known methods: freezing and fixing/embedding aredenaturing either for the morphology of the tissues (freezing method) orfor the molecular structures (fixing/embedding method), thus hinderingthe recognition of the molecules by the specific antibodies, althoughcertain fixing/embedding methods, using especially formaldehyde asfixative, permit denatured proteins to be recognized with the aid of alimited number of antibodies (antiactin, antivimentin . . . ) capable ofrecognizing denatured proteins.

The inherent limitations with the fixing/embedding methods result, onthe one hand, from chemical denaturations induced by the fixativesthemselves, by the dehydrating agents as well as by the organic solventsmiscible with paraffin; on the other hand, thermal denaturations occurduring insertion in the liquid paraffin at 60° C.

The use of a resin with a low melting point (37° C.) avoids the problemsof thermal denaturing.

A solution for minimizing the chemical denaturations consists ofrestricting, at maximum, the number of denaturing substances (alcohols,solvents, . . . ) and using, for the steps preceding embedding,compounds that are completely miscible with embedding resin.

In the following Table 1, the denaturing properties of known methods arecompared with the method of the invention.

TABLE I Comparison of denaturing properties of various methodsMorphological Chemical Method Denaturations Denaturations Freezingstrong weak Fixing/embedding negligible strong with paraffin Acetone +resin weak to average weak to average Method of the negligiblenegligible invention

Examples of Denaturations

The attached figures are given to illustrate the quality ofdeterminations carried out after treatment with the method of theinvention, by comparison with other techniques.

FIGS. 1A to 1C are sections, obtained by freezing, of a tissue of amouse's spleen, with immunohistochemical marking by a biotinylatedanti-B7.2 (GL1) monoclonal antibody, with revelation by alkalinephosphatase.

FIGS. 2A and 2B are similar views, of the red pulp of spleen, afterfixing/dehydration with the acetone and embedding with polyoxyethylenebis(stearate) resin.

FIGS. 3A to 3C are similar views, after treatment according to theinvention.

Denaturations induced by the method of cutting frozen samples are of amechanical nature: crushing, elongation and tearing of the tissue (FIGS.1A and 1B). They are reinforced by the difficulty of making the sectionssufficiently fine in order to be able to locate, with precision, themarkings obtained by the immunohistochemical treatment (FIG. 1C).

In particular:

FIG. 1A is a view at low magnification (4×), the positive cells for themarker B7.2 appear darker,

FIG. 1B is a view at intermediate magnification (25×) that shows “holes”resulting from a tear during cutting,

FIG. 1C is a view at high magnification (100×) showing that it is notpossible to associate the marking with a precise structure.

As mentioned above, the method for fixing with acetone and embeddingwith resin still has disadvantages with respect to the morphological andchemical preservation. FIGS. 2 and 3 show the morphological disordersand compare then with the results obtained with the method involvingfixing with zinc salts.

In particular:

FIG. 2A is a view at intermediate magnification (40×); the largeartefactual spaces between the cells are noted which result from thedilatation of the tissue during fixing;

FIG. 2B is a view at high magnification (100×); the nuclei of the cellsare observed as being darker, but the red blood cells can be scarcelydistinguished.

FIGS. 3A and 3C illustrate perfectly the possibilities of the techniqueof the invention. These results could not be obtained with the freezingmethod (FIG. 1), nor with the method for fixing/dehydrating with acetone(FIG. 2).

In particular:

FIG. 3A is a view at low magnification (4×); one observes specificmarking (darker parts) of dendritic cells (marking with an N418monoclonal antibody, revealing with alkaline phosphatase). Acounter-coloration to the green of the methyl allows one to locate themorphological structures, the nodules of the white pulp appearingclearer, and the red pulp darker,

FIG. 3B is a view at medium magnification (40×) of the dendritic cells(darkest) in the white pulp in contact with the lymphocytes whose nucleiappear clearer. The absence of morphological deformation as well as thepossibility of visualizing each cell is noted,

FIG. 3C is a greatly magnified view (100×) of the red pulp. In additionto the dendritic cells (darker halations in the Figure), one can clearlydistinguish: on the left and halfway up, a large cell of themegacaryocyte type; in the centre, in the lower part, a venous sinus inwhich red corpuscles can be seen, and throughout the slide, the nucleiof all of the nucleated cells.

In addition to the advantage of a perfect preservation of themorphological structures, the method for fixing/dehydrating withzinc/acetone salts, associated with embedding at a physiologicaltemperature of 37° C., offers the possibility of detecting a greaternumber of molecules on sections with the aid of monoclonal antibodies.Table II gives an overview of these possibilities by comparing them withother methods.

TABLE II Comparison of the immunohistological results obtained with thevarious methods Fixing/ Recognized embedding Zinc salts + moleculesFreezing with Acetone + acetone + (antigens) Antibodies method paraffinresin resin B220 +++ − +++ +++ MHC-C12 14.4.4 +++ − +++ +++ CD11b Mac-1+++ − +++ +++ CD11c N418 +++ − +++ +++ IgD LOMD6 +++ − +++ +++ NK 5E6 ++NT NT +++ CD3 7D6 ++ − − +++ CD4 RM4-5 +++ − − +++ CD8 53-6.7 ++ − − +++Vβ8 F23.1 ++ NT NT +++ Vβ6 +/− NT NT +++ B7.2 GL-1 + − +/− +++ DEC-205NLDC-145 + − +/− +++ I1-2 S4B6 +/++ NT − +++ I1-4 11B11 +/++ NT − +++INFγ DB1 +/++ NT − +++ Galβ1-4Gal PNA ++ +++ +++ +++ Note: NT = nottested

In the preceding, polyoxyethylene bis(stearate) resin refers to acomposition containing this compound to which the desired quantity isadded (from 0 to 20% by weight overall, based on 100% by weight of thecomposition) of hexadecanol and/or diethylene glycol distearate, to makeit more solid at room temperature. As indicated, the polyoxyethylenebis(stearate) alone is nevertheless sufficiently solid at a temperatureof 10-12° C. to make it possible to obtain the desired fine sections.

In the same way, although the invention was described with only thisresin, it goes without saying that it can be replaced by any resinhaving similar properties, namely complete miscibility with acetone, amelting temperature not higher than 37-40° C., and sufficient solidityat normal temperature, to enable one to obtain the fine tissue sectionsin laboratory conditions.

Finally, the invention concerns a process for preparing the histologicalsections that enables one to obtain said histological sections accordingto the method of the invention.

We claim:
 1. A method for fixing and embedding a tissue for histologicalpreparations, comprising: fixing the tissue in a liquid fixativecomprising at least one soluble zinc salt, in an aqueous buffersolution, dehydrating the fixed tissue in a liquid consistingessentially of acetone, embedding and infiltrating the fixed anddehydrated tissue, at a temperature not greater than 40° C., with amolten resin which has a melting temperature of not greater than 40° C.which is soluble in any proportion in acetone.
 2. A method according toclaim 1, wherein said embedding and infiltrating step is performed at atemperature not greater than 37° C.
 3. A method according to claim 2,wherein said resin comprises polyoxyethylene bis(stearate).
 4. A methodaccording to claim 1, wherein said resin comprises 80% to 100% by weightof a polyoxyethylene bis(stearate) resin, and 0 to 20% by weight of atleast one compound selected from the group consisting of hexadecanol anddiethylene glycol distearate.